1. Field of the Invention
The invention relates generally to intracellular metabolism and catabolism, and more specifically to a nitric oxide (NO) sensor comprising the N-terminal cysteine amino acid residue sequence motif of a polypeptide, which is subject to oxidation by NO, to methods of identifying agents that modulate the activity of the arginylation branch of the N-end rule pathway, to methods of modulating protein degradation in a cell via the arginylation branch of the N-end rule pathway, and to methods of ameliorating physiological and/or pathological conditions associated with N-end rule pathway-mediated arginylation.
2. Background Information
The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue (1-4; citations can be found following the Examples). The corresponding ubiquitin (Ub)-dependent proteolytic pathway, called the N-end rule pathway, recognizes a set of degradation signals (degrons) that includes the signals called N-degrons. An N-degron includes a destabilizing N-terminal residue of a protein and an internal Lys residue, which is the site of formation of a protein-linked poly-Ub chain. The N-end rule has a hierarchic structure. N-terminal Asn and Gln are tertiary destabilizing residues that function through their deamidation, by N-terminal amidohydrolases, to yield the secondary destabilizing residues Asp and Glu. The activity of N-terminal Asp and Glu requires their conjugation, by ATE1-encoded isoforms of Arg-tRNA-protein transferase (R-transferase), to Arg, a primary destabilizing residues (11, 12). N-terminal arginylated Glu and Asp are recognized by the E3 Ub ligases of the N-end rule pathway. A polyubiquitinylated substrate is processively degraded by the 26S proteasome. The known functions of the N-end rule pathway include the control of peptide import (through conditional degradation of the import repressor), the maintenance of chromosome stability (through degradation of a conditionally produced cohesin fragment), and the regulation of apoptosis (through degradation of a caspase-processed inhibitor of apoptosis), as well as regulation of meiosis, cardiovascular development, and leaf senescence in plants.
Nitric oxide (NO) is produced in eukaryotes primarily by NO synthases. This compound and its derivatives play a role, as either stressors or regulators, in a vast range of functions, including cardiovascular homeostasis, immunity, neurotransmission, ion conductance, glycolysis, apoptosis, and many other processes. Biological effects of NO are mediated by its covalent modifications of proteins, either of their prosthetic groups or amino acid residues, particularly Cys and Tyr. The reactivity of these residues toward NO is modulated by sequence contexts of these residues in a protein. NO converts Cys residues into S-nitrosothiols, a process that can involve oxygen (O2) or its derivatives. S-nitrosylation modulates protein functions either directly or after additional (often oxygen-dependent) chemical transformations that yield oxidized Cys derivatives such as Cys-sulphinic acid (CysO2H) or Cys-sulphonic acid (CysO3H).
In mammals, the set of N-end rule's destabilizing residues that function through arginylation includes Asp, Glu, and Cys, the latter of which is a stabilizing (unarginylated) residue in the yeast Saccharomyces cerevisiae . An N-degron is produced from pre-N-degron through a proteolytic cleavage. Methionine aminopeptidases (MetAPs), which remove Met from the N-termini of newly formed proteins, act if, and only if, the residue at position 2, to be made N-terminal after cleavage, has a small enough side chain (2, and refs. therein). Consequently, among the 13 destabilizing residues of the mammalian N-end rule, only Cys can be exposed at the N-terminus of a nascent protein through the cleavage by MetAPs. Note that any destabilizing residue, including Cys, can be made N-terminal through internal cleavages of proteins by other intracellular proteases such as separases, caspases, and calpains. Previous work showed that Cys at position 2 of the in vivo-arginylated RGS4 protein was CysO3H, rather than Cys, suggesting that oxidation of N-terminal Cys may precede arginylation, and may be required for it. However, the factors involved in the oxidation of cysteine by NO have not been described.